Controlled sample environment for analytical devices

ABSTRACT

Apparatus for examining a sample by microscopy, spectroscopy or crystallography under controlled environmental conditions. The apparatus comprises a sample chamber ( 1 ) which is fed by a gas stream ( 19 ) having a known vapor content, which is generated by mixing two gas streams ( 18  &amp;  16 ), one substantially saturated in a volatile substance ( 18 ) and one substantially free of the volatile substance ( 16 ), in a controlled manner. The temperature of the apparatus, and particularly of the sample chamber ( 1 ), is accurately controlled and regulated by temperature controller ( 7 ).

This application is the National Phase of International ApplicationPCT/GB01/02403, filed May 30, 2001, which designated the U.S. andclaimed priority to Great Britain Application No. 0013261.3 filed May31, 2000.

The present invention relates to apparatus for providing a controlledsample environment for use in conjunction with a microscope, a photonspectroscopic device, an X-ray diffractometer or other analyticaldevice.

Many analytical methods across a range of scientific disciplines arecarried out with the sample in an atmosphere which may not be beneficialto the sample and lead to unreliable analytical results. A chemicalmight degrade in the presence of oxygen or water vapour in theatmosphere or a biological system, e.g. a cell culture, might require aspecific environment in order to maintain its integrity. Chemicaldegradation or loss of integrity of a biological system is known to takeplace, in adverse conditions, over the course of an analyticalexperiment or between experiments which is disadvantageous where severalsets of results are required.

Many organic and inorganic materials are known to react or changechemical and/or morphological states in the presence or absence ofspecific water vapour concentrations. Consequently, the correctcharacterisation of such materials must occur under precisely controlledenvironmental conditions. An analytical system where the environment iscontrolled in terms of temperature and atmosphere (or vapour content) isdesirable. Chemists wishing to make real-time observations, e.g. byinfra-red spectroscopy, of a chemical-reaction mediated by atmosphericcomposition such as air oxidation or hydrogenation under controlledconditions would benefit from such a system.

It is frequently desirable to observe biological cells, for a variety ofpurposes, for extended periods of time. Such experiments may includetime lapse photography where the precise conditions applied to thesystem may be important, but with use of a normal microscope leads todegradation of the system or the requirement that the biological sampleis returned, e.g. to an incubator, between observations. The problemsfaced here are even more pronounced for preparing video footage of abiological event through a suitably adapted microscope.

The provision of particular physical conditions for the opticalmicroscope and other analytical methods has been sought as is evidencedby several of the following patents.

U.S. Pat. No. 4,301,252 relates to a biological incubator for use inconjunction with an optical microscope such that a controlled atmosphereis provided. In particular, the invention permits observation of cellculture (or other biological medium) at physiological conditions. Thetemperature can be held constant and the sample chamber humidified. Thesmall volume of the sample chamber allows for rapid attainment ofdesired atmospheric conditions. The apparatus consists of a miniatureincubator suitable for the placing of a standard petri-dish or culturedish, a hollow fibre purging tube sandwiched between two porous watersaturated pads provides humidified gas to the chamber, which gas escapesthrough the observation hole in the base of the incubator. Heating meansprovided to the incubator consists of e.g. a warm water jacket inintimate contact with the external wall of the incubator. A temperaturesensing means, such as a thermocouple, is provided in the interior ofthe incubator.

U.S. Pat. No. 4,707,086 relates to an assembly which can provide a widerange of temperatures and pressures to the sample cell of an opticalmicroscope to facilitate transient thermodynamic studies. The assemblyconsists of a sample chamber containing optical windows, said chamberhaving protruding into it means for fixing and/or monitoring thetemperature and pressure within the chamber. The chamber is locatedwithin an outer envelope within which is provided means for heating andcooling the chamber. The apparatus is particularly applicable to thestudy of behaviour of crystalline materials placed under pressure infield of geology or study of fluids. More generally, it is applicable tostudies of the effects of pressure and temperature variations in solidstate physics, chemistry and biology.

U.S. Pat. No. 5,257,128 relates to a microscope stage assembly providingcontrol of thermal and fluid environment of samples whilst undersimultaneous observation. The apparatus allows continuous observationand movement/rotation of the sample. The apparatus comprises a samplechamber with an inlet and outlet for fluid supplied from a reservoir forcontrolling the fluid environment, a temperature control means forregulating the temperature in the range −100° C. to 100° C. and opticalwindows for microscopic observation of the sample. This apparatus hasparticular application to studies of cryopreservation of cells and otherbiological materials.

U.S. Pat. No. 4,336,329 relates to apparatus for use in the treatment ofbiological materials, particularly cultivation of biological materials(i.e. a type of incubator) and to a method of using such apparatus. Theatmosphere may be controlled such that the incubator approximatesdifferent conditions in vivo. The apparatus comprises a cultivatingspace fed by a gas (air, oxygen, nitrogen and/or carbon dioxide) inletand a second inlet for steam. The gas streams enter the incubatingchamber via a bypass duct, which may temporarily be closed off from themain incubating space to allow mixing of the two streams. Thecultivating space may be sealed off and heated and cooled as desired.

DE 9414990 U relates to an air conditioning module in which water issprayed into an air stream to create a humidified air stream. Thehumidified air stream can then be mixed with a main air stream, whichpasses a heating element and is then blown from a ventilating zone intothe space in which effective climate control is required.

WO 00/25129 relates to apparatus and a method for inverse gaschromatography. The method includes, for example, the use of a carrierfluid of known vapour content, wherein the vapour is a probe materialfor use in the chromatographic separation.

Although, some of the prior art systems have limited control of theenvironment in terms of temperature or pressure of the sample chamber,or enable the maintenance of high humidity for the study of cellcultures, there is no provision for accurate control of temperature andgaseous composition within a sample chamber of an analytical device.

The present invention provides apparatus for examining a sample bymicroscopy, spectroscopy or crystallography in a controlled environmentcomprising a vapour of a volatile substance, wherein the apparatuscomprises a sample chamber, means for controlling the temperature of thesample chamber, a gas inlet to the sample chamber and a gas outlet fromthe sample chamber, means for generating a first gas streamsubstantially free of the volatile substance, means for generating asecond gas stream substantially saturated by the volatile substance,means for mixing the first and second gas streams in a controlled ratioto generate a third gas stream having a desired content of the volatilesubstance and means for passing the third gas stream to the gas inlet ofthe sample chamber, to provide the controlled environment having thesaid desired content of the volatile substance.

Preferably, there is also provided means for maintaining the apparatus,and particularly, at least the sample chamber, the third gas stream, themeans for mixing the first and second gas streams and the second gasstream at substantially the same temperature.

The invention further provides a sample block for examining a sample bymicroscopy, spectroscopy or crystallography in a controlled environmentcomprising a vapour of a volatile substance, wherein the sample blockcomprises a sample chamber, a gas inlet to the sample chamber and a gasoutlet from the sample chamber, a conduit for containing a first gasstream substantially free of the volatile substance, a chamber forcontaining a liquid reservoir of the volatile substance, the chamberdefines a vapour space above the liquid reservoir, an outlet from thevapour space of the reservoir for passage of a second gas streamsubstantially saturated by the volatile substance, means for mixing thefirst and second gas streams to generate a third gas stream and meansfor feeding the third gas stream to the gas inlet to the sample chamber.

The means for controlling the temperature of the sample chamber maycomprise a temperature controller, a peltier heat pump and a fluidcirculation pump.

It is a preferred feature of the invention that the temperature of thesample chamber can be controlled to within 0.5° C. and more preferablyto within 0.1° C. It is also preferable that the apparatus andparticularly the sample chamber, the second gas stream, the means formixing the first and second gas streams and optionally at least aportion of the first gas stream and the means for generating the secondgas stream may be maintained at the same temperature to +/−0.5° C., andmore preferably to +/−0.1° C.

It is also preferred that the mixing means for generating a third gasstream is such as to enable control of the relative vapour pressure ofthe volatile substance in the third gas stream to within 2.5% and morepreferably to within 1% of a desired relative vapour pressure.

The volatile substance is preferably water, but may be an organicliquid. The first gas stream is preferably a gas which is generallyused, such as dry air, nitrogen, argon or helium.

In one embodiment, the first gas stream is air, the volatile substanceis water and the second gas stream is air saturated with water vapour.The third gas stream is air with a desired proportion of water vapour,from 0% to 100% humidity.

The concentration of volatile substance in the third gas stream andprovided to the sample chamber is controlled by the relative supplies ofthe first gas stream and the source gas provided to the reservoir.

In a particularly preferred embodiment, the sample chamber is locatedwithin a thermally conducting sample block. Such a thermal conductingsample block may be a monolithic block of metal e.g. aluminium, brass orstainless steel.

The second gas stream may be generated within the sample block by meansof passing a source gas through a reservoir of the volatile substance,which is located in a chamber within the sample block for containingsaid reservoir of the volatile substance, to generate a gassubstantially saturated by the volatile substance, said gas exiting saidchamber through an outlet from the gas space of the reservoir. Theinvention may provide a means, located within the sample block, forfeeding the second gas stream to the mixing means. Optionally, theflowpath for the second gas stream is contained entirely within thesample block.

The apparatus of the invention may optionally further comprise a sensorfor measuring the vapour content of the atmosphere. The sensor may bee.g. a relative humidity sensor.

The apparatus may optionally contain windows above and below the samplechamber to allow e.g. observation, transmission of radiation and/orillumination of the sample. The windows are preferably double or tripleglazed to prevent heat loss from the sample chamber and condensation ofthe volatile substance from the atmosphere and may be transparent to thetype of radiation being used to prove the sample e.g. in the case ofspectroscopy and X-ray diffraction and may be chemically resistant tovapours of the volatile substance.

The apparatus of the present invention may further comprise a controlmodule, said control module comprising at least a means for regulatingtemperature and a means for regulating gas flow and optionally a powersupply. The apparatus may further be integrated with a suitablyprogrammed computer.

The apparatus of the present invention may interface with an analyticaldevice, such as a microscope, a photon spectroscopic device or an X-raydiffractometer. The apparatus of the present invention may therefore beused to study physical and chemical changes in a solid or liquid sampledue to the amount of vapour (or humidity) surrounding the sample at aparticular temperature.

The microscope may be, for example, an infra-red microscope or,preferably, an optical microscope.

A preferred embodiment of the invention will now be described withreference to the accompanying drawings. In the drawings:—

FIG. 1 shows a schematic representation of the system in combinationwith a control module and computer.

FIG. 2 shows of a schematic of the relationship of the system whenintegrated with a control module and a computer.

With reference to FIG. 1, the apparatus of the present inventioncomprises a sample chamber (1) located within a sample block (3), ameans for regulating the temperature of the sample chamber (1)comprising a temperature sensor (5), integral with the sample block (3)and sample chamber (1), and a temperature controller (7) located withina control module (9). The sample block (3) is heated and cooled by meansof a Peltier heat pump (11) and an even distribution of temperaturethroughout the sample block (3) is provided by a continuous fluidcirculation channel (13) and a fluid circulation pump (15), all locatedwithin sample block (3).

The fluid in continuous fluid circulation channel (13) may be, forexample, water or silicone oil. The fluid circulation pump (15) mayrecirculate the fluid several times per minute to ensure a fast responseand an even temperature distribution throughout sample block (3).

A first gas stream (16), substantially free of a volatile substancecontained in reservoir chamber (17), is mixed with a second gas stream(18), substantially saturated with the volatile substance, in acontrolled ratio to form a third gas stream (19).

Sample chamber (1) is fed with a third gas stream (19) through an inlet(20) to the sample chamber and is vented through an outlet (21).

The second gas stream (18) is generated by feeding a controlled flow ofgas from a gas source (23) through a liquid reservoir (25) of thevolatile substance located in the reservoir chamber (17) within sampleblock (3). The vapour space (27) of reservoir (25) contains a gassaturated with the volatile substance which provides the second gasstream (18) by escaping through an outlet from the vapour space (30).

The ratio of the first (16) and second (18) gas streams present in thethird gas stream (19) is controlled by Mass Flow Controllers (31) and(33) for controlling the mass flow of source gas providing the first(16) and second (18) gas streams respectively. The mass flow controllers(31 & 33) are located in the control module (9) and fed from a gassource (23).

A computer (41) may be connected to the control module (9) for automatedtemperature and gas composition of the apparatus. The concentration ofthe volatile substance in third gas stream (19) is defined by theproportions of the first gas stream (16) and second gas stream (18) andthe temperature of the sample block (3).

1. Apparatus for examining a sample by microscopy, spectroscopy or crystallography in a controlled environment comprising a vapour of a volatile substance, wherein the apparatus comprises: a sample chamber; means for controlling the temperature of the sample chamber; a gas inlet to the sample chamber and a gas outlet from the sample chamber; means for generating a first gas stream substantially free of the volatile substance; means for generating a second gas stream substantially saturated by the volatile substance; means for mixing the first and second gas streams in a controlled ratio to generate a third gas stream having a desired content of the volatile substance; and means for passing the third gas stream to the gas inlet of the sample chamber, to provide the controlled environment having the said desired content of the volatile substance.
 2. Apparatus as claimed in claim 1, which further comprises means for maintaining: the sample chamber, the third gas stream, the means for mixing the first and second gas streams, and the second gas stream, at substantially the same temperature.
 3. Apparatus as claimed in claim 2, wherein the temperature maintaining means is such as to maintain the said temperature to within +/−0.1° C. of a desired temperature.
 4. Apparatus as claimed in claim 1, for examining a sample by microscopy, which further comprises an optical microscope.
 5. Apparatus as claimed in claim 1, wherein the volatile substance is water.
 6. Apparatus as claimed in claim 1, wherein the first gas stream is dry air.
 7. Apparatus as claimed in claim 1, wherein the second gas stream is humidified air.
 8. Apparatus as claimed in claim 1, wherein the sample chamber is located within a thermally conducting sample block.
 9. Apparatus as claimed in claim 8 wherein the mixing means is located in the thermally conducting sample block.
 10. Apparatus as claimed in claims 8 or 9 wherein the means for generating a second gas stream comprises: a chamber located in the sample block for containing a reservoir of the volatile substance; and means for passing a gas flow through the liquid reservoir in the chamber to provide the second gas stream substantially saturated by the volatile substance.
 11. Apparatus as claimed in claims 8 or 9, wherein the second gas stream is contained entirely within the sample block.
 12. Apparatus as claimed in claim 1, further comprising a sensor for measuring the content of the volatile substance in the third gas stream.
 13. Apparatus as claimed in claim 1, wherein the sample chamber further comprises a viewing window for the said examination.
 14. Apparatus as claimed in claim 1, further comprising a control module, said control module comprising: means for regulating temperature; and means for regulating gas flow.
 15. Apparatus as claimed in claim 10, wherein the second gas stream is contained entirely within the sample block.
 16. Apparatus as claimed in claim 14, further comprising a computer suitably programmed to operate with the apparatus.
 17. A sample block for examining a sample by microscopy, spectroscopy or crystallography in a controlled environment comprising a vapour of a volatile substance, wherein the sample block comprises: a sample chamber; a gas inlet to the sample chamber and a gas outlet from the sample chamber; a conduit for containing a first gas stream substantially free of the volatile substance; a chamber for containing a liquid reservoir of the volatile substance, the chamber defines a vapour space above the liquid reservoir; an outlet from the vapour space of the reservoir for passage of a second gas stream substantially saturated by the volatile substance; means for mixing the first and second gas streams to generate a third gas stream; and means for feeding the third gas stream to the gas inlet to the sample chamber.
 18. A sample block as claimed in claim 16, which includes means for feeding a gas through the liquid reservoir to form the second gas stream.
 19. A sample block as claimed in claim 16, further comprising a sensor for measuring the composition of the third gas stream.
 20. Apparatus for examining a sample by microscopy, spectroscopy or crystallography in a controlled environment comprising a vapour of a volatile substance, wherein the apparatus comprises: a sample chamber; a temperature controller for controlling the temperature of the sample chamber; a gas inlet to the sample chamber and a gas outlet from the sample chamber; a gas stream generator that generates a first gas stream substantially free of the volatile substance; a gas stream generator that generates a second gas stream substantially saturated by the volatile substance; a gas mixer that mixes the first and second gas streams in a controlled ratio to generate a third gas stream having a desired content of the volatile substance; said third gas stream being directed to the gas inlet of the sample chamber to provide the controlled environment having the said desired content of the volatile substance. 